Formalin-fixed and paraffin-embedded human hepatocarcinoma tissue reacted with Autophagy GABARAP Antibody , which was peroxidase-conjugated to the secondary antibody, followed by DAB staining. This data demonstrates the use of this antibody for immunohistochemistry; clinical relevance has not been evaluated.
GABARAP antibody was tested in mouse MEFs and mouse motor neurons. Cells were fixed in 3% PFA and permeabilized with 100% methanol. Antibodies used were anti-GABARAP (1:500, labeled with anti-rabbit 488 Alexa) and Covance anti-Tuj1 (1:1000, labeled with anti-mouse 647).
Fluorescent image of U251 cells stained with GABARAP antibody. U251 cells were treated with Chloroquine (50 mu M,16h), then fixed with 4% PFA (20 min), permeabilized with Triton X-100 (0.2%, 30 min). Cells were then incubated GABARAP primary antibody (1:500, 2 h at room temperature). For secondary antibody, Alexa Fluor 488 conjugated donkey anti-rabbit antibody (green) was used (1:1000, 1h). Nuclei were counterstained with Hoechst 33342 (blue) (10 ug/ml, 5 min). GABARAP immunoreactivity is localized to autophagic vacuoles in the cytoplasm of U251 cells.
Rabbit anti-GABARAP 1:1000 dilution in 5% skimmed milk powder. Secondary antibody goat anti-rabbit-HRP ECL detection 30 sec on X-ray film.
Western blot of GABARAP (arrow) using purified antibody. 293 cell lysates (2 ug/lane) either nontransfected (Lane 1) or transiently transfected with the GABARAP gene (Lane 2) (Origene Technologies).